Ghrelin Receptor Signaling in Health and Disease: A Biased View

Abstract

As allosteric complexes, G protein-coupled receptors (GPCRs) respond to extracellular stimuli and pleiotropically couple to intracellular transducers to elicit signaling pathway-dependent effects in a process known as biased signaling or functional selectivity. One such GPCR, the ghrelin receptor (GHSR_1a), plays a crucial role in restoring and maintaining metabolic homeostasis during disrupted energy balance. Thus, pharmacological modulation of GHSR_1a bias could offer a promising strategy to treat several metabolism-based disorders. Here, we summarize the current evidence supporting GHSR_1a functional selectivity in vivo and highlight recent structural data. We propose that precise determinations of GHSR_1a molecular pharmacology and pathway-specific physiological effects will enable discovery of GHSR_1a drugs with tailored signaling profiles and thereby, provide safer and more effective treatments for metabolic diseases

Section I: G Protein-Coupled Receptors (GPCRs).

GPCRs (see Glossary) comprise the largest receptor family in the human genome [1]. They are homologous plasma membrane spanning proteins composed of an extracellular N-terminus, seven transmembrane segments connected by alternating intracellular and extracellular loops, and an intracellular C-terminus (Figure 1). GPCRs transduce extracellular chemical, optical, and mechanical information into intracellular signaling cascades for nearly every physiological system. Their common structures support a broad array of drug-accessible binding determinants, including principal orthosteric binding pockets that activate signaling and accessory allosteric sites that modulate signal strength. Because of their membrane accessibility and biological importance, GPCRs are the most common targets for FDA-approved drugs [2]. In drug discovery, pharmacologists have recently become very interested in new classes of orthosteric and allosteric compounds that can selectively bias GPCR conformations and signaling in a manner distinct from the endogenous (natural) ligands, thereby favoring atypical signaling with differential physiological outcomes — a process referred to as functional selectivity or biased signaling. Although few such compounds have reached the clinical stage, their far reaching therapeutic implications has prompted directed searches for functional selectivity GPCR pharmacophores with tailored signaling profiles to ameliorate specific disease pathways. The current belief is that, in most instances, functionally selective GPCR therapies can provide superior therapeutic efficacy and/or safer side effect profiles relative to unbiased/balanced pharmaceutical standards-of-care.

Figure 1. GHSR_1a conformational states and signaling modes.

(a) Monomeric, ghrelin-bound GHSR_1a elicits signaling through both G protein (G_q/11, G_i/o, and G_12/13, particularly G_q/11) and Gβγ/GRK2-dependent βarr (particularly βarr2) pathways. (b) Pro148Ala mutation (orange) in ICL2 constrains GHSR_1a G protein signaling conformation(s), whereas (c) Leu149G mutation (purple) constrains the receptor to βarr2-favoring conformation(s). The GHSR_1a may form (d) homo- (dark green-light green) or (e) GPCR heterodimers (e.g., D2 dopamine receptor [D2R]) (dark green-yellow) that may influence ligand binding and/or biased signaling.

Origins of GPCR Signaling Bias.

GPCR agonists can potentially signal through different, cell-specific pathways by selectively engaging available G proteins or β-arrestins (βarrs) (Fig. 1A). G proteins form heterotrimeric complexes of single α, β, and γ subunits. In humans, there are 20 α, 6 β, and 12 γ isoforms, enabling a remarkable number of αβγ combinations. Though limited in number, the two non-visual arrestins βarr1 and βarr2 (a.k.a. arrestins 2–3) are profligate scaffolding proteins that compensate for their ‘lack of diversity’ by binding a multitude of intracellular signaling molecules and forming multimeric, regulatory protein complexes. A comprehensive review of G protein and arrestin structure and function can be found elsewhere [3, 4]. G proteins and βarrs compete both temporally and spatially by occupying overlapping intracellular receptor sites in intracellular loops (ICLs) and C-tail regions of GPCRs. G protein-coupled receptor kinase (GRK) phosphorylation of these regions in agonist-occupied or constitutively active receptor conformations enhances βarr binding, hindering G protein coupling and leading to receptor desensitization, clathrin-mediated endocytosis, and endosomal trafficking of the βarr-coupled receptor [5]. Additionally, the extracellular domains (ECDs) of GPCRs, including residues within the extracellular loops (ECLs), extracellular vestibule, and superficial TM regions, serve as ‘hotspots’ for ligand binding, allosteric modulation, and biased signaling by fine-tuning receptor conformational states [6, 7]. Through allosteric coupling within a ternary complex of ligand-receptor-transducer [8], ligand binding in the ECD stabilizes receptor conformation(s) that are transmitted to transducer-coupling motifs on the intracellular face of the receptor to influence signal transduction [9].

Section II: Ghrelin Receptor Signaling & Functional Selectivity.

The ghrelin receptor (GHSR_1a) is a rhodopsin-like, Class A GPCR and the cognate, high-affinity receptor [10] for the peptide agonist ghrelin [11] and the peptide antagonist/inverse agonist LEAP-2 (liver-expressed anti-microbial protein-2) [12]. In this review, we will focus primarily on the molecular pharmacology of ghrelin, a 28 amino acid hormone synthesized predominantly in gastric enteroendocrine cells [13] and released via sympathetic adrenergic activation in response to caloric restriction or environmental stress [14, 15]. Ghrelin peptide exists in one of two forms: unacetylated (desacyl-ghrelin) or acylated at Ser³ with an octanoyl fatty acid (acyl-ghrelin) via a reaction catalyzed by the endoplasmic reticulum-associated enzyme, ghrelin-O-acyltransferase (GOAT) [16]. Circulating acyl-ghrelin levels tightly correlate with energy states (e.g., obesity, fed-fasted, starvation) [17], despite being the minority species in circulation [18]. By utilizing the octanoyl moiety as a direct interaction determinant, acyl-ghrelin binds the GHSR_1a with nanomolar affinity. In contrast, desacyl-ghrelin constitutes the majority of circulating ghrelin [18], but the absence of an octanoyl moiety reduces GHSR_1a affinity by several orders of magnitude, thereby rendering sub-micromolar concentrations of desacyl-ghrelin incapable of initiating receptor signaling under standard physiological conditions [11, 19]. However, desacyl-ghrelin does elicit physiological effects, suggesting mechanism(s) involving yet-to-be-identified receptor target(s) [20].

GHSR_1a Functional Selectivity.

The GHSR_1a couples preferentially to G_q/11, but it is also capable of coupling to the G_i/o and G_12/13 families [21–23]. Thus, the GHSR_1a can exhibit signaling bias for specific Gα subunits resulting from either the intrinsic properties of the ligand/receptor interaction (ligand bias) or the relative availability of different Gα subunits in the biological system (system bias). GHSR_1a coupling to βarr elicits cellular or physiological responses that can be distinguished from G protein-dependent outcomes [24] (Figure 2). A complete bias between G_q/11 and βarr signaling has been demonstrated using two contiguous point mutations within intracellular loop 2 (ICL2), +6 (Pro148) or +7 (Leu149) [25, 26] amino acids downstream of the highly conserved, transducer-coupling (E)DRY motif (Fig. 1B--C).C). Therefore, stabilizing GHSR_1a conformations selective for a subset of signaling pathways should, in principle, also be attainable pharmacologically.

Figure 2. Evidence-based in vivo GHSR_1a functional selectivity and potential therapeutic applications of biased GHSR_1a ligands.

(Top) Simplified model of GHSR_1a functional selectivity and downstream physiological/behavioral outcomes based on existing preclinical literature. (Bottom) Hypothesized disease targets and corresponding biased (agonist or allosteric modulator) drug candidates based on mechanisms shown in the Top image (black text: stronger evidence; grey text: weaker or indirect evidence) Ghrelin peptide (red circle), GHSR_1a (dark green), Gq heterotrimer (Gα_q subunit-blue, guanine triphosphate (GTP)-red, Gβ subunit (orange), Gγ subunit (light green), βarr2 (blue oval), allosteric modulator (blue circle).

Constitutive Activity and the Extracellular Loops (ECLs).

The GHSR_1a intrinsically exhibits high (~50% ligand max) [27] and physiologically-relevant constitutive activity. Several missense mutations in ECL2, including some naturally-occurring (e.g., Ala204Glu and Phe279Leu), abolish GHSR_1a constitutive activity and are associated with idiopathic short stature and abnormal body weight in humans [28]. Interestingly, the Ala204Glu mutation markedly reduces ghrelin-induced βarr2 signaling efficacy, but does not appreciably affect ghrelin binding or ghrelin-mediated G protein signaling [29]. Mice expressing the homologous ECL2 point mutation (Ala203Glu) display reduced ghrelin-stimulated food intake and GH secretion, age-dependent reductions in body weight, body length, and bone length, and diminished GH and blood glucose levels in response to caloric restriction [28], similar to effects seen with GHSR KO mice [30]. These findings, coupled with mutagenesis studies [23, 29], support that ECL2 is a critical determinant of both ligand-dependent and -independent GHSR_1a bias, as well as GHSR_1a-mediated metabolic function.

Pathway Biasing of GHSR_1a Signaling by Protein-Protein Interactions.

Several in vitro studies support that GHSR_1a dimerizes with itself (homodimer) and other GPCRs (heterodimer) (Fig. 1D), including the D1 and D2 dopamine (DA), serotonin 2C, cannabinoid receptor type 1, orexin, melanocortin 3 [31], GHSR_1b splice variant [32], and several others. In each case, the GHSR_1a and its dimer partner exhibit some degree of altered expression, signaling, or trafficking relative to the monomeric protomers alone, supporting that these interactions may occur via canonical allosteric mechanisms. Studies also suggest that GHSR_1a ligands display different binding affinities and signaling efficacies for the orthosteric (R1) protomer and allosteric (R2) protomer that compose a homodimer. The simultaneous occupation of R1 by ghrelin and R2 by an ago-allosteric agonist — a compound possessing intrinsic efficacy and allosteric activity [33] — can increase ghrelin signaling efficacy [34]. In contrast, ghrelin acts similarly in a purified receptor system of GHSR_1a homodimers versus monomers [32]. Together these studies support that GHSR_1a homodimerization modulates receptor function in a ligand-specific manner. In addition, the GHSR_1a also binds the melanocortin 2 receptor accessory protein (MRAP2) via an interaction required for ghrelin’s orexigenic [35] and insulinostatic [36] effects. MRAP2, which is expressed in GHSR_1a-positive hypothalamic-pituitary and pancreatic cells, blunts GHSR_1a constitutive activity and biases ghrelin-stimulated signaling towards G_q over βarr [37], supporting it as key regulator GHSR_1a system bias.

Section III. GHSR_1a Expression, Physiology, and Disease.

Ghrelin Brain Penetrance and Brain GHSR_1a Distribution.

Despite an appreciable increase in our understanding of ghrelin physiology over the past two decades, important mechanistic questions remain related to central ghrelin function during various metabolic states. These questions exist, in large part, because of unusually high GHSR_1a constitutive activity, GHSR_1a proximity to circumventricular organs (CVOs) and fenestrated capillaries, and the disparate pharmacological properties of acyl- and desacyl-ghrelin. A comprehensive review of these questions can be found elsewhere [38, 39]. In brief, acyl-ghrelin synthesis via GOAT in brain is minimal [40, 41] and unlike desacyl-ghrelin, is poorly blood-brain barrier (BBB) permeable during normal physiological states. Nonetheless, acyl-ghrelin can still readily enter brain regions proximal to CVOs comprised of fenestrated capillaries, such as the olfactory bulb (OB), brain stem, and hypothalamic nuclei; whereas, GHSR_1a distal from CVOs have restricted access to physiological levels of acyl-ghrelin. Identifying GHSR_1a-expressing regions with restrictive molecular diffusion relies on well-developed measurement techniques, such as systemically bioavailable radioligand- and fluorescence-based ghrelin tracers. Outside of areas supporting transluminal diffusion (i.e., BBB-restricted), GHSR_1a-mediated effects likely rely on ligand-independent mechanisms, such as GHSR_1a constitutive activity and/or homo/hetero-oligomerization. Although ligand-independent GHSR_1a signaling is difficult to assess in vivo, studies using inverse agonists in mice support a role for GHSR_1a constitutive activity in energy state-dependent feeding and body weight regulation [42]. Collectively, the current evidence supports that acyl-ghrelin brain penetrance is species- [43], metabolic state- [44], and region-dependent [45], with ghrelin brain penetrance enhanced during negative energy balance (e.g., fasting, caloric restriction) and region-dependence associated with GHSR_1a proximity to CVOs (e.g., median eminence, area postrema). Accordingly, GHSR_1a in the hypothalamus and brain stem can be activated locally by circulating ghrelin via direct entry through proximal fenestrated capillaries, thereby enabling rapid and dynamic responsiveness to changes in energy demand. Additionally, acyl-ghrelin can traverse the blood-cerebrospinal fluid (CSF) barrier via hypothalamic tanycyte uptake [46], albeit with low efficiency. Conversely, GHSR_1a-expressing regions protected by the BBB may mediate ghrelin-independent effects on a distinct time scale during metabolic homeostasis and become accessible only when energy balance is significantly perturbed.

Hypothalamus and Pituitary.

The GHSR_1a is widely expressed both peripherally and centrally [34]. In the CNS, it is expressed most highly within the agouti-related protein (AgRP)-positive neurons of the hypothalamic arcuate nucleus (ARC) [47]. In ARC AgRP neurons, the GHSR_1a regulates homeostatic feeding, adiposity, and energy metabolism [48]. Hypothalamic GHSR_1a is also abundant within growth hormone-releasing hormone (GHRH) neurons [49]; wherein, GHSR_1a tonically and phasically stimulates growth hormone (GH) secretion from anterior pituitary somatotrophs [11], which sits outside the CNS.

During negative energy balance, GHSR_1a transcription and ghrelin peptide synthesis/secretion are increased in the ARC [42] and periphery [50] to restore metabolic homeostasis via orexigenic, adipogenic, anabolic, and hyperglycemic responses that increase food-seeking behavior, decrease energy expenditure, mitigate bone or muscle loss, and maintain blood glucose levels via insulinostatic and gluconeogenic effects [51]. During positive energy balance, such as obesity, ghrelin resistance occurs by dampening these processes in a reversible manner [52]. Ghrelin-mediated feeding and GH secretion requires G_q activation [22, 53] and the latter effect supports anabolism and hyperglycemia by inducing insulin-like growth factor-1 (IGF-1) release from liver, hepatic gluconeogenesis, and insulin resistance [54].

The Limbic System and Dopaminergic Midbrain Nuclei.

The GHSR_1a is also abundant in extra-hypothalamic, limbic regions, including DA midbrain nuclei such as the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) [55]. Dopaminergic VTA (VTA^DA) neurons project to the ventral striatum to regulate motivation, reinforcement, and the incentive salience of palatable foods and drugs of abuse [56]. Dopaminergic SNc (SNc^DA) neurons project to the dorsal striatum to regulate motor coordination and habit formation [57]. In both regions, the GHSR_1a increases DA neuron firing and elevates downstream striatal DA release, thereby increasing locomotion and reward-seeking [55]. Genetic or pharmacological blockade of the GHSR_1a in dopaminergic neurons blunts reward behavior [58], and βarr2 in DA neurons is required for GHSR_1a-dependent reward behavior [25, 59]. In SNc^DA neurons, the GHSR_1a provides neuroprotection downstream of adenosine monophosphate kinase (AMPK) phosphorylation, likely via G_q-dependent Ca²⁺ mobilization [60]. Indeed, ghrelin-induced pAMPK activation in hypothalamus is lost in G protein-biasing MRAP2 KO mice [37]. Consistent with well-established effects of pAMPK, GHSR_1a activation enhances several cell survival by reducing apoptosis, increasing autophagy and mitophagy, promoting mitochondrial biogenesis, and suppressing reactive oxygen species (ROS) generation [61]. Collectively, these effects make GHSR_1a activation protective against the loss of SNc^DA neurons and striatal DA tone observed in Parkinson’s disease (PD) [62].

Using a transgenic eGFP knockin mouse under control of the GHSR promoter [63], Zigman and colleagues showed that GHSR-eGFP expression was distributed throughout several additional, higher-order limbic brain regions that lack significant GHSR mRNA expression, including the prefrontal cortex, insular cortex, amygdala, and hippocampus. GHSR_1a in these areas likely account, at least in part, for the antidepressant, anxiolytic, pro-cognitive effects of GHSR_1a activation. Collectively, these energy state-dependent affective responses, in conjunction with hypothalamic and DA-dependent appetitive drive, are thought to support food-seeking by diminishing perceived predatory risk (anti-depressive, anxiolytic) [51] and enhancing spatial navigation and context-dependent remote memory (pro-cognitive) within nutrient-containing environments [64].

Pancreas.

In peripheral metabolic tissue, GHSR_1a is expressed in pancreatic islets — particularly somatostatin (SST)-positive δ-cells [65, 66], and to a lesser extent in insulin-containing β-cells [67, 68] and glucagon-containing α-cells [69]. Here, the GHSR_1a may exert both paracrine and autocrine insulinostatic effects via G_q/Ca²⁺- [65] and G_i/o- dependent [70] mechanisms, respectively. Local pancreatic ghrelin is secreted from ε-cells following stimulation by β1Rs under conditions of increased sympathetic drive (e.g., fasting) [14, 15]. Accordingly, GHSR_1a-mediated insulinostasis causes acute hyperglycemia and reduced glucose tolerance by reducing β-cell glucose-stimulated insulin secretion (GSIS) [71], thereby supporting brain glucose utilization during periods of caloric deficiency. Conversely, GHSR_1a antagonists and inverse agonists are insulinotropic and improve glucose tolerance [72, 73]. To maintain euglycemia during starvation, GHSR_1a-dependent increases in blood glucose are potentiated by GH-mediated gluconeogenesis [74]. In the fed state, hormonal and metabolite satiety factors, including LEAP-2 [75], glucagon-like peptide-1 [GLP-1] [76], and blood glucose coordinately blunt plasma ghrelin levels [77].

Gastrointestinal Tract.

GHSR_1a activation stimulates gastric emptying in both healthy individuals and those with diabetic gastroparesis [78, 79]. The prokinetic effect is lost in GHSR KO mice and may rely on the βarr, G_i/o, and/or G₁₃ rather than G_q or G₁₂ based on pharmacological evidence [22]. A synthetic GHSR_1a agonist, relamorelin, increases gastric emptying and reduces GI symptoms in both type 1 and type 2 diabetes, prompting clinical interest [77].

Cardiovascular System.

GHSR_1a is vasodilatory in heart and peripheral vasculature, anti-hypertensive, negatively inotropic, and cardioprotective after cardiac damage [77]. Interestingly, ghrelin does not increase intracellular Ca²⁺ levels in vascular smooth muscle cells and it inhibits angiotensin II-induced Ca²⁺ mobilization in rat aorta in an adenylyl cyclase-dependent manner [80]. Thus, the GHSR_1a may regulate cardiovascular function, at least in part, via a G_q-independent mechanism.

Section IV. A Structural Basis of GHSR_1a Pharmacology and New and Better Drugs.

Drug development is undergoing a revolution precipitated by advances in GPCR structural determinations and computational processing power. In prior decades, drug discovery efforts have relied on high-throughput screening programs over days-to-weeks to test compound libraries of ~100,000+ molecules. Whereas, current in silico screening of virtual compound libraries containing ~10,000,000+ compounds can be accomplished more rapidly using commercially-available software, desktop computers, and published GPCR structural models. Within the last five years, several homology or structural models of the ligand-bound GHSR_1a were generated from nuclear magnetic resonance (NMR), X-ray crystallography, and most recently, cryogenic electron microscopy (cryo-EM). These models collectively provide molecular maps that detail ligand binding-associated receptor conformations for a variety of signaling states. A structure-driven approach enables better predictive modeling of how other compounds may stabilize these conformations and thereby, permit more fine-tuned drug discovery programs.

Nuclear magnetic resonance (NMR)-based models of the GHSR_1a.

Bender et al. employed solid-state NMR, virtual modeling, and receptor mutagenesis to show that acyl-ghrelin residues, Ser³ and Phe⁴, were required for a stable interaction with the GHSR_1a orthosteric binding pocket and furthermore, that acyl-ghrelin Pro⁷ and His⁹ were critical for stabilizing an extended, α-helical binding mode spanning the GHSR_1a-ECD (including TMVI, TMVII, and ECL3 [81]). That same year, Ferre et al. used solution-state NMR and modeling and suggested, in contrast, that the hydrophobic core of acyl-ghrelin (residues 8–18) lacked an α-helical arrangement [82]. Both models, however, indicated that the N-terminal Gly¹ cap of acyl-ghrelin stabilizes binding deep within the GHSR_1a orthosteric pocket by an ionic interaction with Glu124^TMIII, a conserved ‘anchor’ residue that otherwise forms a constitutive ionic lock with Arg283^TMVI of unoccupied (apo-) GHSR_1a [83]. Both studies also indicate multiple interactions between ghrelin and a hydrophobic cluster in TMVI and TMVII of the receptor. Mutations to either Glu124^TMIII, Arg283^TMVI, or the hydrophobic cluster prevent GHSR_1a ligand activation [81]. These studies provide a structural basis to show the minimal acyl-ghrelin GHSR_1a binding fragment is composed of only its first five N-terminal residues [84].

Applications of Ghrelin Receptor Modeling to Drug Development.

We utilized a ghrelin-bound GHSR_1a model generated by Bender et al. as a structural template to computationally study binding poses of the small-molecule GHSR_1a ligand, NCATS-SM8864 (a.k.a. N8279) [23]. NCATS-SM8864 is a selective GHSR_1a agonist with G_q bias compared to other G proteins (G_s, G_i/o, G_12/13) and βarr2. In mice, systemic NCATS-SM8864 administration is readily brain penetrant, an uncommon property for GHSR_1a agonists [85]. Molecular docking simulations of NCATS-SM8864 into the apo-and ghrelin-bound GHSR_1a using commercially available software (Schrӧdinger) and supporting mutagenesis experiments demonstrated that the GHSR_1a-ECD, particularly ECL2, is a critical determinant of NCATS-SM8864 signaling relative to ghrelin. Importantly, the GHSR_1a ECL2 ‘caps’ the extracellular vestibule, which regulates ligand entry to and egress from the ligand binding pocket and influences signaling bias [7]. Therefore, structural modulation of the GHSR_1a-ECD may provide an efficient strategy to discover additional, functionally selective GHSR_1a ligands.

GHSR_1a Inactive Structure by X-Ray Crystallography.

GHSR_1a Inactive Structure by X-Ray Crystallography. GPCRs are large, flexible proteins that remain difficult to crystalize because they reside in mixed aqueous-lipid environments. To overcome these inherent difficulties, biochemists have developed several techniques using antibodies and inserted peptide sequences to stabilize GPCRs. The first human GPCR X-ray crystal structure was solved in 2007 [86], and the first GHSR_1a X-ray crystal structure was not solved until 2020 [83]. The Kojima group crystalized human GHSR_1a bound to a neutral antagonist at a resolution of 3.3 Å (PDB: 6KO5) that modeled an inactive, non-signaling conformation. The structure revealed a bifurcated orthosteric ligand binding pocket consisting of two cavities separated by a large gap involving TMVI and TMVII. The gap, or crevasse, contains the hydrophobic, phenylalanine (Phe)-rich residue cluster recognized by NMR studies and is capped by ECL2. Cavity I and cavity II are demarcated by the salt bridge between Glu124^TMIII and Arg283^TMVI (Figure 3). Further structural analysis and mutagenesis suggested that the Phe cluster (Phe279^TMVI, Phe309^TMVII, and Phe312^TMVII) in the TMVI/TMVII ‘crevasse’ accommodates the ghrelin Ser³-octanoyl and thus, is required for receptor activation [83].

Figure 3. Orthosteric ligand binding pocket of the GHSR_1a.

Cavity I and II of the human GHSR_1a depicted with a (A) snake plot and (B) surface densities of the ghrelin-bound, G_q-coupled receptor (Wang et al., 2021). A bifurcating salt bridge (broken upon agonist binding) between Glu124^TMII (side chain in red) and Arg283^TMVI (side chain in blue) demarcates two distinct cavities that accommodate the N-terminal peptide moiety (Cavity I, blue) and Ser³-acyl moieties (Cavity II, red) of ghrelin. Note, the central β-sheet of ECL2 caps Cavity I, whereas the C-terminal helix of ECL2 caps Cavity II.

Models of the GHSR_1a Active Structure and Cryo-EM.

In 2021, Wang et al. [87] used recent advances in cryo-EM technology to solve the structure of a human GHSR_1a-G_q (chimera) complex. Cryo-EM is rapidly becoming the method-of-choice to image GPCRs at high resolution. In contrast to the conformational uniformity required of X-ray crystals, cryo-EM reconstructs the most probable conformations of receptor/ligand/transducer complexes by imaging millions of sample complexes on EM grids. The Wang et al. study solved GHSR_1a structures bound to ghrelin or the small peptide agonist growth hormone releasing peptide-6 (GHRP-6) at a resolution of 2.9 Å and 3.2 Å [87] (PDB: 7F9Y and 7F9Z), respectively. In the ligand-bound active conformation, the acyl- moiety of ghrelin occupied cavity II, rather than the TMVI/TMVII ‘crevasse’ purported from the crystal structure [83]. The acyl- moiety stabilizes the N-terminal peptide fragment of ghrelin (Gly¹ to Pro⁷) deep within the TM bundle of cavity I. Polar and charge-rich amino acids of the ghrelin peptide (Glu⁸ to Arg¹⁵) extend upward toward the ECD in an α-helical conformation, as described by the NMR-based, Bender et al. model [81]. The data further provided structural evidence that the minimal, signaling-competent ghrelin peptide fragment requires only its N-terminus (amino acids 1–5). The interactions between ghrelin and cavity I residues are preferred to the constitutive ionic lock formed by Glu124^TMIII and Arg283^TMIV in the apo-GHSR_1a state. Displacement of this salt bridge reorients the Arg283^TMIV side chain and drives contrasting outward and inward movements of intracellular TMVI and extracellular TMVII, respectively [87]. Indeed, these are the conserved, hallmark conformational changes of GPCR activation [84].

GHSR_1a-G_q Activation Mechanism.

G protein coupling at GHSR_1a-ICLs is enabled by agonist-induced rearrangement of the Glu124^TMIII-Arg283^TMIV salt bridge, which reorients the Arg283^TMIV side chain and stimulates a series of conformational changes in conserved “micro-switch” motifs, thereby transitioning the receptor from the inactive to the active state. These changes include, a disruption of the ionic lock between the (E)/DRY^TMII/TMIII motif and TMVI, an engagement of a CWxP motif rotamer switch at residues in TMVI, and rearrangements of NPxxY^TMVII and transmembrane PIF motifs. Notably, these changes were observed in the ghrelin- and 6-GHRP-bound agonist states [87], but not in the antagonist-bound state [83], despite all three ligands displaying overlapping occupation of the orthosteric binding pocket. In summary, GHSR_1a activation requires disruption of the Glu124^TMIII-Arg283^TMVI ionic lock to drive intracellular outward movement of TMVI, which exposes the G protein coupling interface and enables signal transduction.

GHSR_1a-G_i Active Structure.

Shortly after the above studies were published, Liu et al. [88] solved both the cryo-EM GHSR_1a-G_i structure bound to ghrelin and the small-molecule, unbiased growth hormone secretagogue ibutamoren (MK-0677) at a resolution of 2.7 Å (PDB: 7NA7 and 7NA8). Similar to the inactive [83] and G_q-coupled [87] GHSR_1a conformations, the ghrelin-bound GHSR_1a-G_i structure revealed a bifurcating Glu124^TMIII-Arg283^TMVI salt bridge and supported that the acyl- moiety occupies Cavity II. Furthermore, ghrelin’s N-terminal peptide moiety made comparable polar and hydrophobic interactions with residues previously identified in the G_q-coupled GHSR_1a structure and showed an ionic interaction between ghrelin-Gly¹ and Glu124^TMIII. As expected, the agonist-bound, G_i-coupled GHSR_1a exhibits Arg283^TMVI reorientation, intracellular outward movement of TMVI, extracellular inward movement of TMVII, and engagement of micro-switch residues (e.g., rotamer toggle switch residue, Trp276^TMVI). Together with the other structural studies discussed, these findings support a common GHSR_1a activation mechanism. Interestingly, Liu et al. observed cryo-EM densities of the ghrelin-bound GHSR_1a that corresponded to cholesterol-enriched domains, which they show act as a positive allosteric modulator (PAM) to enhance ghrelin binding and signaling [88]. Others also reported recently that membrane lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2), can allosterically modulate the GHSR_1a [89].

GHSR_1a-G_o Active Structure.

The most recent cryo-EM GHSR_1a structure published (to-date) solved the ghrelin-bound, G_o-coupled GHSR_1a conformation. This structure was obtained in parallel with a crystallographic determination of the GHSR_1a bound to the inverse agonist PF-05190457, and each were solved a resolution of <3 Å (PDB: 7W2Z and 7F83) [90]. The ghrelin-bound, G_o-coupled GHSR_1a showed conformational changes consistent with those observed in the G_q-coupled structure [87]. Conversely, GHSR_1a bound to PF-05190457 — a drug that has been evaluated in clinical trials for the treatment of alcohol use disorder (AUD) and type 2 diabetes (T2D) [91, 92] — showed diametrically opposite conformational changes relative to agonist-bound structures; namely, a marked inward movement of TMVI at its intracellular end and outward movements of both TMVI and TMVII at their extracellular ends [90]. In contrast, the ghrelin-bound, G_o-coupled GHSR_1a displayed micro-switch residue engagement, an intracellular outward movement of TMVI, and an inward extracellular movement of TMVII [90], similar to the G_q- [87] and G_i-coupled [88] structures. The authors note that ghrelin’s acyl- moiety occupies a moderately different orientation relative to observations of the G_q study [88]. This suggests that differences of acyl-moiety positioning within the binding pocket may explain, in part, ghrelin’s differential intrinsic efficacy between different G protein subfamilies (e.g., G_q versus G_i/o) [23]. Specifically, the acyl- moiety points towards the interface of TMV and TMIV in G_q [87] and G_i [88] structures, whereas it is oriented directly towards TMV in the G_o-coupled complex.

Basic Mechanisms Underlying GHSR_1a Activation.

The GHSR_1a structural data highlight the key conformational changes GPCRs undergo upon ligand binding (described below and in Figure 4A). Collectively, these studies provide molecular scale signatures for each major pharmacological category: antagonist [83], peptide and small-molecule agonist [87, 88, 90], and inverse agonist [90]. Thus, these findings provide a roadmap for developing GHSR_1a ligands across multiple drug classes. In summary, the key findings from these recent GHSR_1a structures support the following (as depicted in Figure 4):

Figure 4. Distinct conformational differences between the agonist (ghrelin)-, antagonist-, and inverse agonist-bound GHSR_1a coupled to different G proteins.

(a) Intracellular outward movement of TMVI (Cα: His258) relative to TMIII (Cα: Ill145) in the ghrelin-bound, G_q-coupled GHSR_1a (green) compared to antagonist-bound (red) and inverse agonist-bound (orange) structures. Dotted lines: Intramolecular (TMVI) distance between the agonist- and inverse agonist-bound GHSR_1a and intermolecular distance between agonist-bound TMVI and TMIII. (b) Extracellular inward movement of TMVII (Cα: Ill297) towards TMVI (Cα: Ser289) in the ghrelin-bound, G_q-coupled GHSR_1a (green) compared to antagonist-bound (red) and inverse agonist-bound (orange) structures, as well as the inward movement and ‘capping’ (purple) of ECL2 in the agonist-bound receptor. Arrows = β-sheets. (c) Position and orientation of the octanoyl group on Ser³ of acyl-ghrelin within the orthosteric ligand binding pocket. Ghrelin-bound, G_q- (green), G_i- (teal), and G_o- (blue) coupled GHSR_1a structures. (d) GHSR_1a-Gα (C-terminus) coupling interface for the ghrelin-bound, G_q- (green), G_i- (teal), and G_o- (blue) coupled structures. Representative GHSR_1a residues (Thr78^TMII, Arg141^TMIII, Lys329^H8) proximal to the Gα coupling interface make distinct interactions or occupy distinct space in a Gα subunit-dependent manner.

Key Findings from Recent Structural Studies

• Signaling is initiated by pronounced conformational changes in TMVI, TMVII, and ECL2.

• Agonists drive the outward movement (Δ12.1 Å) of intracellular TMVI relative to TMIII, exposing the G protein binding pocket and enabling coupling (Figure 4A).

• Conversely, inverse agonists drive inward movement (Δ8.3 Å) of intracellular TMVI towards TMIII, occluding the G protein binding interface (Figure 4A).

• The antagonist-bound GHSR_1a is relatively static.

• Receptor conformational distinctions between ligand classes are mediated primary by TMVI.

• The inward movement (Δ6.5 Å) of extracellular TMVII towards TMVI, and ECL2 capping of the ligand binding pocket (Figure 4B), enhance ligand affinity by positive allosteric coupling.

• The ECL2 cap is comprised of a β-sheet spanning residues Glu197 through Pro200 (Figure 4B), two residues important for signaling of NCATS-SM8864, a G_q-biased GHSR_1a ligand [23].

• Inverse agonists drive an outward movement (Δ8.9 Å) of TMVII relative to TMVI and prevent ECL2 capping, thereby occluding transducer coupling and expanding the extracellular vestibule (Figure 4B).

• Subtle changes in the position and orientation of ghrelin’s acyl-moiety (Figure 4C) and the G protein coupling interface (Figure 4D) may explain, in part, differences in ghrelin signaling efficacy observed for different G proteins (G_q versus G_i/o) [23].

Concluding Remarks.

The mechanistic insights obtained from high resolution GHSR_1a structural studies will expedite the design and discovery of new, functionally selective GHSR_1a pharmacotherapeutics. By minimizing side effects, toxicity, and/or tachyphylaxis, functionally selective drugs should be particularly useful for treating chronic diseases. A tailored drug design strategy will be especially useful for age-related conditions and metabolic diseases of disrupted energy balance by narrowly directing efficacy towards pathological drivers, and by avoiding or diminishing on- and off-target perturbations of GHSR_1a-dependent homeostatic systems. Disorders that could benefit from GHSR_1a pharmacotherapy will eventually be categorized by the key signaling pathway disrupting the underlying physiology, thereby enabling functionally selective pharmacotherapeutics to be identified rationally (see Outstanding Questions). For example, drugs that selectively stimulate G_q and attenuate βarr signaling may be efficacious as treatments for anorexia or cachexia via stimulation of hypothalamic and pituitary G_q signaling, neurodegenerative disease via G_q/Ca²⁺/AMPK-dependent neuroprotection, and/or drug addiction via inhibition of βarr-dependent reward learning. Conversely, drugs that selectively increase βarr and attenuate G_q signaling may be efficacious as treatments for obesity via inhibition of hypothalamic G_q signaling, gastroparesis by increasing gastric emptying/motility, or cardiovascular disease via G_q-independent cardioprotection (Figure 3). Advancing a class of biased/functionally selective GHSR_1a therapeutics into the clinic will require a concerted effort to (a) obtain ligand-stabilized GHSR_1a structures in biased conformations, (b) develop GHSR_1a animal models with complete pathway bias to employ in translationally-relevant physiological assays, and (c) evaluate systems bias by exploiting transcript/proteomic datasets to identify GHSR_1a bias-modulating proteins and transducer expression levels in target cell-/tissue-types (see Outstanding Questions). Collectively, these approaches will elucidate the mechanisms that mediate GHSR_1adependent metabolic disease and thereby, enable successful drug discovery efforts that capitalize on the GHSR_1a functional selectivity.

OUTSTANDING QUESTIONS.

Structural

• How distinguishable is the βarr-coupled GHSR_1a conformation from G protein (G_q, G_i/o)-coupled conformations?

• Do biased GHSR_1a ligands stabilize GHSR_1a conformations distinct from ghrelin-driven states? If so, does this involve alterations in ECL2 capping of the orthosteric binding pocket?

Biochemical

• Are GHSR_1a-mediated feeding, GH release, insulinostasis, and neuroprotection entirely βarr-independent?

• How can systems bias be leveraged to design or develop functionally selective GHSR_1a compounds, and could this enable cell- or tissue-specific drug action for personalized medicine?

Clinical:

• Will biased, genetically-engineered animal models be superior tools over biased ligands to decipher the contribution GHSR_1a and GPCR functional selectivity to clinically-relevant, physiological outcomes?

• Will functionally selective drugs improve personalized medicine? For example, relative to unbiased/balanced ligands, can biased GHSR_1a drugs be developed to treat neurological disorders selectively without producing peripherally-mediated, metabolic side effects?

• As a disrupting technology that democratizes drug discovery, the question appears not to be if, but how quickly in silico screening will replace current, industry-based methods of high-throughput screening. How will this contribute to the advancement of novel, functionally selective GHSR_1a and GPCR drugs to the clinic?

HIGHLIGHTS.

• In a G protein-coupled conformation, acyl-ghrelin occupies two cavities of the GHSR_1a demarcated by an inter-transmembrane (TMIII-TMVI) salt bridge, with its octanoyl group in one cavity and its N-terminal peptide moiety in the other. Only minor conformational differences exist between the ghrelin-bound, G_q-coupled GHSR_1a and the G_i/o-coupled receptor.

• Agonist-stimulated GHSR_1a activation rearranges the TMIII-TMVI salt bridge, produces intracellular outward movement of TMVI, extracellular inward movement of TMVII, and ECL2 capping of the orthosteric binding pocket. ECL2 is a key determinant of signaling and bias.

• The GHSR_1a adopts conformational states capable of dramatic G_q and β-arrestin signaling bias and pathway-dependent physiological effects. More evidence is needed to define β-arrestin-specific outcomes.

• Biased GHSR_1a drugs can target chronic illness associated with metabolism- and age-related disease, including obesity, type 2 diabetes, cachexia, neurodegenerative disorders, and cardiovascular disease.

GLOSSARY

Ago-allosteric agonist

a ligand that activates the receptor in both the absence (apo-) and modulates signaling of the natural ligand

Agonist

a ligand that stabilizes an active receptor conformation and elicits signal transduction above basal levels

Allosteric Coupling

the reciprocal process whereby conformational changes affect the conformational state of another, topographically distinct protein within a multimeric complex (e.g., ternary complex of ligand-receptor-transducer)

Allosteric ligand

a ligand that binds distinct, non-overlapping receptor site(s) relative to the endogenous (natural) ligand

Allosteric modulation

the ability of an allosteric ligand to augment or inhibit the signaling of another ligand when co-occupying the receptor

Antagonist

a ligand that binds the receptor and prevent conformational shifts, thereby preventing receptor signaling from going above or below basal levels

Biased signaling or functional selectivity

signal transduction through only a subset of a receptor’s possible intracellular signaling pathways

Constitutive activity

receptor-mediated signaling that occurs independent of ligand binding (basal activity)

Cryogenic electron microscopy (cryo-EM)

an experimental technique using electron microcopy to determine the three-dimensional molecular structure of proteins stabilized in cryogenically frozen grids

ECL2 Cap

a portion of ECL2 that sits over top the ligand binding pocket in the transducer-coupled conformational state

G protein-coupled receptor kinase (GRK)

a family of kinases that phosphorylate the C-terminal tail of GPCRs to increase βarr recruitment and binding to the receptor

G protein-coupled receptor (GPCR)

a large family of integral membrane proteins that control myriad physiological processes and are the most common pharmaceutical drug target

Growth hormone secretagogue

a stimulus that increased growth hormone secretion

Heterodimer

a physical interaction between two different proteins

Homodimer

a physical interaction between two identical proteins

Insulinostatic

a stimulus that inhibits insulin secretion

Insulinotropic

a stimulus that increases insulin secretion

Inverse Agonist

a ligand that stabilizes an inactive conformation and elicits signal transduction below basal levels

Ligand bias

the process whereby a ligand stabilizes a receptor conformation that disproportionally affects (activates or inhibits) signaling through only a subset of all possible signal transduction pathways

Negative energy balance

when energy expenditure exceeds energy intake (e.g., fasting)

Neuroprotection

the promotion of neuronal cell survival or health following stress or damage

Nuclear magnetic resonance (NMR)

an experimental technique that measures the electromagnetic signatures of nuclei in magnetic fields by techniques such as isotopic spin labeling to determine the three-dimensional molecular structure and dynamics of proteins

Orexigenic

a stimulus that increases food intake

Orthosteric ligand

a ligand that binds receptor site(s) shared by the endogenous (natural) ligand

Positive energy balance

when energy intake exceeds energy expenditure (e.g., obesity)

System bias

biased signaling/functional selectivity resulting from differential expression of transducers or accessory/modulatory proteins in a given biological system

Unbiased/balanced ligands

ligands that evoke signaling equally through all of a receptor’s possible intracellular signaling pathways

X-ray crystallography

an experimental technique using X-ray diffraction to determine the three-dimensional molecular structure of proteins stabilized in crystals